SecA Localization and SecA-Dependent Secretion Occursat New Division Septa in Group B StreptococcusSara Brega1,2, Elise Caliot1,2, Patrick Trieu-Cuot1,2, Shaynoor Dramsi1,2*
1 Institut Pasteur, Unite de Biologie des Bacteries Pathogenes a Gram positif, Paris, France, 2 CNRS, ERL 3526, Paris, France
Abstract
Exported proteins of Streptococcus agalactiae (GBS), which include proteins localized to the bacterial surface or secreted intothe extracellular environment, are key players for commensal and pathogenic interactions in the mammalian host. Theseproteins are transported across the cytoplasmic membrane via the general SecA secretory pathway and those containingthe so-called LPXTG sorting motif are covalently attached to the peptidoglycan by sortase A. How SecA, sortase A, andLPXTG proteins are spatially distributed in GBS is not known. In the close relative Streptococcus pyogenes, it was shown thatpresence of the YSIRKG/S motif (literally YSIRKX3Gx2S) in the signal peptide (SP) constitutes the targeting information forsecretion at the septum. Here, using conventional and deconvolution immunofluorescence analyses, we have studied inGBS strain NEM316 the localization of SecA, SrtA, and the secreted protein Bsp whose signal peptide contains a canonicalYSIRKG/S motif (YSLRKykfGlaS). Replacing the SP of Bsp with four other SPs containing or not the YSIRKG/S motif did notalter the localized secretion of Bsp at the equatorial ring. Our results indicate that secretion and cell wall-anchoringmachineries are localized at the division septum. Cell wall- anchored proteins displayed polar (PilB, Gbs0791), punctuate(CspA) or uniform distribution (Alp2) on the bacterial surface. De novo secretion of Gbs0791 following trypsin treatmentindicates that it is secreted at the septum, then redistributed along the lateral sides, and finally accumulated to the poles.We conclude that the 6YSIRK SP rule driving compartimentalized secretion is not true in S. agalactiae.
Citation: Brega S, Caliot E, Trieu-Cuot P, Dramsi S (2013) SecA Localization and SecA-Dependent Secretion Occurs at New Division Septa in Group BStreptococcus. PLoS ONE 8(6): e65832. doi:10.1371/journal.pone.0065832
Editor: Eliane Namie Miyaji, Instituto Butantan, Brazil
Received February 5, 2013; Accepted April 29, 2013; Published June 7, 2013
Copyright: � 2013 Brega et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by grant ANR-06-PATHO-001-01 from the Agence Nationale de la Recherche ‘‘ERA-NET PathoGenoMics’’ (to P. T.-C.)and by the Institut Pasteur. The authors also acknowledge funding from the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence‘‘Integrative Biology of Emerging Infectious Diseases’’ (grant nuANR-10-LABX-62-IBEID). The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Introduction
Streptococcus agalactiae (also known as Group B Streptococcus,
GBS) is an extracellular gram-positive encapsulated bacterium
that colonizes the adult gastrointestinal tract and vaginal flora of
15–30% of the healthy women [1]. When transmitted from a
‘carrier’ mother to newborn infants, this opportunistic pathogen
can provoke invasive neonatal diseases, including sepsis, pneumo-
nia, and meningitis [2]. Besides neonatal infections, S. agalactiae is
an emerging pathogen in the elderly and in individuals suffering
from underlying diseases [3]. Exported proteins, which include
proteins localized to the bacterial surface or secreted into the
extracellular environment, are major determinants of virulence in
S. agalactiae as they play key roles in colonization of host tissues and
control of immune responses [4]. Most bacterial proteins that
translocate across the cytoplasmic membrane possess a N-terminal
signal peptide and are exported to extracellular compartments via
the highly conserved general secretory SecA-YEG pathway. These
secreted proteins are either released into the extracellular milieu or
anchored to the cell wall by the housekeeping class A sortase if
they possess a C-terminal LPXTG cell wall sorting signal
consisting of an LPXTG-like motif followed by a hydrophobic
domain and a positively charged tail [5]. SrtA cleaves the LPXTG
motif and links the carboxyl-group of the threonine to the
peptidoglycan precursor lipid II [6]. The protein-lipid II-linked
product is then incorporated into the mature peptidoglycan (PG)
via the cell wall biosynthesis machinery. In silico analysis of S.
agalactiae NEM316 genome predicts the existence 35 LPXTG
proteins [7] thought to be covalently attached to the cell wall by a
unique SrtA [8].
In the close relative Streptococcus pyogenes, Rosch and Caparon
showed that the SecA motor and secreted proteins specifically
localize to a microdomain named ExPortal which is distal to the
poles [9]. This finding was subsequently challenged by Carlsson et
al. who showed a uniform distribution of SecA in the membrane of
S. pyogenes. Remarkably, Carlsson et al. also found that the signal
peptide of M protein, which contains an YSIRK motif (SP+YSIRK),
directs secretion to the division septum, while that of protein F
(PrtF, also known as SfbI) lacking this motif (SP2YSIRK) targets the
old pole [10]. Recently, Raz et al. confirmed the differential
localization of M and PrtF/SfbI proteins in S. pyogenes and further
provided a dynamic 3D view of protein localization during cell
cycle [11]. The M protein is rapidly anchored at the septum,
simultaneously at the mother and daughter septa. In contrast the
SP2YSIRK SfbI protein accumulates gradually on peripheral
peptidoglycan resulting in a polar distribution. Protein localization
is not perturbed in a SrtA mutant, suggesting that signal sequence
directs localized secretion before cell wall anchoring [11]. In
contrast, impairment of septum assembly results in marked
reduction in the amount of M protein, but not of Sfbl. A
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functional divisome is thus critical in the anchoring of SP+YSIRK-
type proteins and may control their expression/stability [11].
The signal peptide-dependent protein localization rule is also
true in Staphylococcus aureus. SP+YSIRK CWA proteins (protein A,
ClfA, SdrC, SdrD, and FnbpB) were shown to be distributed in a
ring-like manner near the vicinity of the cell division site, whereas
SP2YSIRK proteins (SasA, SasD, SasF, and SasK) were found at
discrete peripheral loci on the bacterial envelope [12]. Recently,
generation of a mCherry reporter system to investigate the spatial
localization of surface proteins in live S. aureus further confirmed
that SP+YSIRK and SP2YSIRK are necessary and sufficient to drive
localization of CWA proteins either to the equator/septum or
pole, respectively [13]. Interestingly, treatment with sub-lethal
concentrations of penicillin shifted the localization of the non-
YSIRK mCherry hybrids from the peripheral cell wall to the
septum in a SrtA-dependent manner. This relocalization may be
due to increased amounts of lipid II precursors at the septum [13].
In the present work, we investigated the subcellular localization
of SecA, SrtA, two secreted proteins (Bsp and CAMP), and four
cell wall-anchored proteins (Alp2, PilB, CspA and Gbs0791) in the
coccoid S. agalactiae using immunofluorescence microscopy. Our
main finding is that SecA and SrtA are both localized at the
division septum suggesting that SecA-dependent secretion and
SrtA-dependent cell wall anchoring are spatially coupled in S.
agalactiae. Thus, the polar distribution of some LPXTG proteins
(e.g. PilB and Gbs0791) likely reflects a post-secretion process
dependent on SrtA.
Results
SecA localizes at equatorial rings and division septa inexponentially growing GBS
Despite its importance to bacterial physiology and virulence, the
subcellular localization of SecA of S. agalactiae has never been
reported. We determined the localization of SecA in exponentially
growing cells using a polyclonal rabbit antibody raised against
SecA of E. coli. We first checked the specificity of this antibody and
showed that it reacts with a single band of the expected size of
about 90 kDa in a Western blot analysis of whole cell extracts of S.
agalactiae strain NEM316 and E. coli strain DH5a as a positive
control (Fig. S1). Immunofluorescence microscopy analysis
revealed a defined localization of SecA at equatorial ring, i.e. a
zone of active peptidoglycan synthesis, in growing streptococcal
chains (Fig. 1A). Quantitative image analysis of 15 different fields
at lower magnification (e.g. Fig. S2) showed that amongst 880
bacteria counted, 797 formed a chain (.2 cocci) with the typical
SecA labeling as shown in Fig. 1 (90%), 81 single cocci displayed a
uniform labeling (9%, Fig. S2), and only 2 (0.2%) were not labeled.
Ovococcal bacteria, like streptococci, divide along the perpendic-
ular axis of a chain and rely on the activity of two finely
coordinated peptidoglycan machineries at the mid-cell that are
dedicated either to cell elongation or septation [14]. Fluorescent-
vancomycin, which binds to peptidoglycan (PG) precursor [15], is
a useful tool to visualize novel sites of PG synthesis on
streptococcal chains [16]. Double fluorescence labeling experi-
ment using both anti-SecA antibody and fluorescent vancomycin
indicates that SecA and vancomycin mostly co-localizes at septal
sites (Fig. 1A). Of note, a few septal sites that potentially
correspond to constriction sites were not labeled with SecA (white
arrows in Fig. 1, last panel). Deconvolution microscopy confirmed
the localization of SecA at equatorial septa in GBS strain NEM316
(Fig. 1B). In contrast, we found that SecA was uniformly
distributed on the bacterial surface of GAS strains (Fig. 2) as
previously published [10].
Extracellular proteins are found at the division septa of S.agalactiae
Bsp (group B secreted protein) is a highly conserved extracel-
lular protein of 65 kDa [17] encoded by gbs1420 in strain
NEM316 genome [7]. This protein exhibits limited similarity
(,25%) to the murein hydrolase lysostaphin and possesses four
SH3 domains and an atypical LPXTG signature lacking the
downstream hydrophobic sequence. In line with this observation,
Bsp protein is found almost exclusively in the culture supernatant
[17]. GBS also secretes another well-characterized 25 kDa pore-
forming toxin known as CAMP factor [18–20]. To investigate the
subcellular distribution of Bsp and CAMP proteins, we made use
of specific polyclonal antibodies against Bsp and CAMP that were
previously used [21,22]. Using deconvolution immunofluorescence
microscopy, we showed that Bsp and CAMP proteins co-localize
with green fluorescent vancomycin at septal sites in strain
NEM316 (Fig. 3). As for SecA labeling, a few division sites that
may correspond to constrictions sites were not labeled with Bsp or
CAMP (white arrows).
Localization of Bsp to the septum does not depend on aYSIRK signal peptide
The signal peptide dependent localization rule defined in GAS
[10,13] and S. aureus [12] specifies that a signal peptide containing
the YSIRK/GS motif directs secretion close to the cell division site
(septum), whereas non-YSIRK signal peptide directs proteins near
the poles. However, this rule is apparently not observed in GBS
(Fig. 3) as Bsp contains a typical YSIRK signal peptide but not the
CAMP protein (Fig. 3C).
To further test this localization rule in GBS, we decided to swap
the signal peptide of Bsp with those of four cell wall-anchored
proteins, two containing the YSIRK motif (Alp2 and CspA) and
two without it (PilB and Gbs0791), and the chimeric genes were
expressed in the low-copy number plasmid pTCV-erm under the
control of the constitutive Tn916 tetM promoter (PtetM). A
schematic representation of theses constructs is shown in Fig. 4A.
We first showed that the various signal peptides allowed the
secretion of Bsp, as shown by dot blot on whole bacteria using
exponential and stationary growing bacteria (Fig. 4B). Western
blotting of supernatant protein extracts revealed that the four
different constructs directed synthesis of a specific band of about
60 kDa corresponding to Bsp, while the control vector did not
(Fig. 4C). This result demonstrates that YSIRK- and non-YSIRK
containing signal peptides enable the secretion of Bsp at similar
levels (Fig. 4C). We next examined by immunofluorescence
microscopy the localization of these four secreted Bsp differing
by their signal peptides (Fig. 5). In exponentially growing bacteria,
all Bsp isoforms were mainly localized at the septum and thus, we
concluded that localization of a secreted protein in S. agalactiae is
independent of the YSIRK motif.
Subcellular localization of SrtA- and SrtA- substrates in S.agalactiae
We next aimed at characterizing the subcellular distribution of
the cell wall anchoring enzyme SrtA and of some SrtA-substrates
for which specific antibodies were available. We first checked the
specificity of the antibody raised against GBS SrtA. Western
blotting showed a band at approximately 30 kDa (SrtA ca MW
27,503.7) in the total protein extracts of NEM316 strain that was
absent in the isogenic DsrtA mutant (not shown). Immunofluores-
cence analysis showed that SrtA, like SecA, localizes at equatorial
rings and division septa in exponentially growing GBS (Fig. 6A).
However, it is worth to mention that SrtA showed a more diverse
Immunolocalization of Surface Proteins in GBS
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Figure 1. Distribution of SecA in S. agalactiae NEM316. Bacteria grown overnight in 10 ml of TH (OD600<2) were diluted to get an initial OD600
of 0.05 (1/40 dilution) and grown at 37uC until OD600 reached 0.5 and re-diluted again in TH (1/10) until reaching an OD600 of 0.5 and diluted again (1/10) before final collection at mid-exponential phase (OD600 of 0.3) to get a homogenous population of exponentially growing cells. Bacteria werepretreated with lysozyme (1 mg/mL final concentration) for 15 min at 37uC and then permeabilized with PBS-Triton X-100 (0.4%) for 5 min at RT,washed twice with PBS and then fixed with PBS containing 3% paraformaldehyde for 15 min at RT. (A) Differential interference contrast (DIC) andimmunofluorescence microscopy (IFM) of bacteria harvested in mid-exponential phase and visualized with rabbit anti-SecA pAb (red) or fluorescentvancomycin (green) plus rabbit anti-SecA pAb (red). White arrows in the last panel indicate potential constriction septa. Image representative of atleast 800 GBS chains analyzed (B) Deconvolution images of sequential z-sections (0.3 mm) of NEM316 cells labeled with rabbit anti-SecA pAbpresented as maximum intensity projections.doi:10.1371/journal.pone.0065832.g001
Figure 2. Distribution of SecA at the surface of S. agalactiae (GBS NEM316) and S. pyogenes (GAS M18). (A) Western blot image showingthat the polyclonal antibody directed against E. coli SecA recognizes a band of approximately 90 kDa in both GAS M18 and GBS NEM316 strains. (B)Conventional immunofluorescence microscopy showing the differential distribution of SecA at the surface of GBS NEM316 versus GAS strain M18collected in exponential and stationary phase of growth. Heterogeneity of SecA distribution was quantified by eye following analysis of randomlyselected fields.doi:10.1371/journal.pone.0065832.g002
Immunolocalization of Surface Proteins in GBS
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distribution than SecA (Fig. S2). We next analyzed the subcellular
distribution of four LPXTG-containing proteins that are anchored
to the cell wall by the SrtA sortase (Fig. 6B). These were PilB, the
major pilin of the PI-2A pilus [23]; Gbs0791, a highly conserved
protein of unknown function (our unpublished data); Alp2, a
major surface protein [24]; and CspA, a surface serine protease
that cleaves human fibrinogen [25] and CXC chemokines [26].
Note that PilB and Gbs0791 do not contain the YSIRK motif in
their signal peptides whereas Alp2 and CspA do possess the
YSIRK motif (Fig. 4). Surprisingly, although the septal localization
of SecA and SrtA machineries in S. agalactiae strongly suggest that
secretion of cell wall-anchored proteins occurs at the septum, we
observed that these cell wall-anchored proteins displayed various
patterns: polar (PilB, Gbs0791), punctuate (CspA) or uniform
distribution (Alp2) on the bacterial surface (Fig. 6B). We interpret
these results as indicating that these proteins were dynamically
redistributed on the bacterial surface after their secretion.
De novo secretion of Gbs0791 occurs at the septumThe redistribution of a polar protein on the GBS surface was
further studied as follows. Since PilB containing pili are highly
resistant to trypsin and proteinase K treatments (data not shown),
we chose to analyze the site of Gbs0791 de novo secretion after
shaving the bacterial surface with trypsin as previously described
[10]. Immunofluorescence analysis showed that Gbs0791 was
rapidly and efficiently removed by trypsin from the bacterial
surface. We observed that this protein re-appeared at septal sites
after 1 h of regeneration, was later redistributed laterally to
recover the entire bacterial surface after 2 h of regeneration, and
finally displayed a preferential polar localization in stationary
phase bacteria (Fig. 7).
Protein localization is strongly altered in a non-capsulated mutant
The capsule of S. agalactiae is an extracellular polysaccharide that
plays a key role in virulence [27]. Immunofluorescence analysis of
SecA distribution in the non-capsulated DcpsE mutant showed a
more diffuse labeling compared to the defined labeling in the
parental strain NEM316 (Fig. 8A). Similarly, localization of the
secreted Bsp and the cell wall-anchored PilB is strongly altered in
the non-capsulated mutant as compared to the parental NEM316
strain (Fig. 8B).
Discussion
In this report, we describe the first subcellular localization of
endogenous unrelated secreted proteins in the coccoid Streptococcus
agalactiae (GBS), as determined by immunofluorescence microsco-
py using specific antibodies. These were the general motor ATPase
SecA of the general secretory pathway, two known secreted
proteins Bsp and CAMP, the cell wall anchoring enzyme SrtA,
and four SrtA-substrates (also known as LPXTG-containing
proteins). Our main finding is that SecA-dependent secretion
and SrtA anchoring machineries are spatially coupled and
localized at the division septum in exponentially growing culture
of S. agalactiae, collected after successive dilutions, to get a
homogeneous population of individual cells (OD600 of 0.3).
We found that Bsp and CAMP, two abundantly secreted
proteins, were also localized at the division septa in exponentially
growing bacteria. Sequence analysis revealed that the Bsp signal
peptides displayed a canonical YSIRK motif whereas the CAMP
signal peptide did not, which in turn suggested that the YSIRK
signal peptide-dependent localization rule did not apply to S.
agalactiae. This rule, first proposed in S. pyogenes [10] and later
Figure 3. Subcellular localization of Bsp and CAMP factor in S. agalactiae NEM316. (A, B) IFM images of bacteria harvested in exponentialphase (OD600 = 0.3) and labeled with specific antibodies directed against Bsp (A) or CAMP factor (B) revealed with AlexaFluor 594- fluorescentsecondary antibody (red). Outline of the cells was visualized by DIC and active zone of peptidoglycan synthesis with fluorescent vancomycin (green).(C) Signal peptides of Bsp and CAMP proteins. The amino acids constituting the YSIRK motif are highlighted in red. Schematic localization ofvancomycin, Bsp, and CAMP factor at non-constricting septa.doi:10.1371/journal.pone.0065832.g003
Immunolocalization of Surface Proteins in GBS
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confirmed in S. aureus [12], stipulates that YSIRK-containing
signal peptide drives the localization of a reporter protein to the
septum whereas non-YSIRK signal peptide targets the reporter
protein to the pole [13]. Intriguingly, mutations in the conserved
YSIRK motif did not change the localization pattern of the
corresponding proteins [10,12], suggesting that this motif is not per
se the localization signal but rather a ‘‘tag’’ for a class of signal
peptides with specific properties. Of note, deletion of SP+YSIRK in
protein A from S. aureus reduced the amount of anchored
polypeptide [28]. Indeed, the SP+YSIRK-mCherry chimera is more
highly expressed than the SP2YSIRK-mCherry chimera [13].
In this work, the GBS protein Bsp was chosen as a secretion
reporter protein and we constructed a set of variants by replacing
its native SP with that of four S. agalactiae cell wall-anchored
proteins, two containing (Alp2 and CspA) and two lacking (PilB
and Gbs0791) the YSIRK motif. To avoid artifacts due to
interferences with the native protein and/or differential expression
levels, all recombinants proteins were similarly expressed from the
same vector into NEM316:Dbsp mutant strain. We first showed
that all tested SP allowed the secretion of Bsp in the culture
supernatant at approximately the same level (Fig. 4). Importantly,
as shown in Fig. 5, the localization pattern is very similar to that of
the native Bsp in WT strain NEM316, with a majority of bacteria
displaying a septal localization of Bsp that appears to be
independent of the signal peptide directing its secretion. It is
worth noting that increasing the amount of Bsp on the bacterial
surface through overexpression was associated with the presence of
a significant increase amount of protein to the lateral sites, but not
with the redistribution of the protein to the poles (data not shown).
Very few localization studies have been performed in S.
agalactiae. In particular, the highly conserved protein Sip
(NEM316 Gbs0031), the group B surface immunogenic protein,
has been found at septal and polar regions by immunogold
electron microscopy [29]. The signal peptide of Sip lacks the
YSIRK motif but the mature secreted protein contains a LysM
domain that might mediate binding to the peptidoglycan. Sip has
been localized both at the cell surface and secreted in the culture
supernatant of all GBS strains tested [29]. How can we explain the
Figure 4. Expression in GBS of Bsp recombinant proteins with structurally unrelated signal peptides. (A) BamHI-NotI PCR fragmentscarrying the ribosome binding site (RBS) and the signal peptides (SP) of 5 SecA-dependent substrates (Bsp, Alp2, Gbs0791, PilB, and CspA) were fusedin frame with a NotI-PstI PCR fragment coding the Bsp protein devoid of its signal peptide (Table S1). The resulting BamHI-PstI fragments were cloneddownstream the constitutive PtetM promoter from the low-copy-number pTCV-erm. The SP and Bsp sequences are indicated in upper-bold italiccharacters and upper-bold characters, respectively. The boxed RP motif in all proteins corresponded to the translation of the two internal codons ofthe NotI restriction site (CGGCCG). All but one SP were predicted with SignalP 4.1 (www.cbs.dtu.dk/services/SignalP/) whereas the remaining (Alp2)was predicted with PrediSi (www.predisi.de). The AA residues in the SP thought to direct localized secretion at the bacterial surface are indicated inred characters. Arrowheads indicate the predicted site of cleavage of the various SP. (B) Analysis of surface display of Bsp recombinant proteins in aDbsp mutant strain by immunoblotting. Whole bacterial cells harvested in exponential (OD600 0.3) or stationary (OD600 1.2) phases were washed,resuspended in phosphate buffer saline to similar density and spotted on nitrocellulose. Membranes were hybridized with specific anti-Bspantibodies or with anti-GBS pAb (loading control). (C) Western blotting analysis of culture supernatants. Proteins were separated on 4–12% gradientTris-acetate Criterion XT SDS-PAGE gel, then transferred onto a nitrocellulose membrane, and detected by immunoblotting with specific anti-Bsp andanti-CAMP antibodies. In (B) and (C), the Dbsp mutant strain harboring pTCV-erm (negative control) or pTCV-erm directing synthesis of recombinantBsp proteins associated with Alp2, Gbs0791, PilB, and CspA signal peptides were used.doi:10.1371/journal.pone.0065832.g004
Immunolocalization of Surface Proteins in GBS
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polar localization of Sip? In streptococci, which divide in parallel
plane along the short axis of the bacterial chain, elongation occurs
faster than division and therefore future septa of daughter cell are
visible in a bacterial chain before cell separation. Therefore, polar
distribution of a protein could result either from an active targeting
to the poles or a passive targeting occurring when the septum turns
into new pole following bacterial division. From our results on the
localization of SecA and other secreted proteins, we hypothesize
that secretion occurs at the septum and protein accumulation at
the poles is the result of long-lived proteins.
In related streptococci, different localization patterns have been
described for both SecA and sortase A (for a recent review, see
[37]). In S. pyogenes, Rosch et al. proposed that SecA colocalizes
with sortase A in a single membranal microdomain termed
ExPortal [9,30]. This domain is enriched in anionic lipids and thus
can be visualized using a dye such as N-nonyl-acridine orange
NAO. However, another study, revealed that SecA is distributed
uniformly [10], a result that we confirmed using our anti-SecA
antibody on GAS M18 and M49 strains (Fig. 2 and data not
shown). Recently, S. pneumoniae SecA and SecY were localized in
the mid-cell region, whereas the sortase A was found over the
surface of cells without discernable pattern [31]. An ExPortal–like
anionic lipid microdomain similar to that described previously in
GAS [32] was detected in GBS NEM316, but it is clearly not
associated with SecA equatorial localization (Fig. S3). This
observation rules out the model of ExPortal in S. agalactiae.
Consistently, we similarly showed that SrtA is localized mainly at
the septum reinforcing the idea of a multi-protein complex
coupling protein secretion, cell wall- anchoring and peptidoglycan
synthesis at the division site. Interestingly, SecA, Bsp, and CAMP
labeling seem to disappear from constricting septa (Fig. 1 and 3),
an observation that needs further investigation using video-
microscopy. SrtA was found mostly at the septum, but also as
discrete foci as described for SecA in Bacillus subtilis [33] or
randomly over the entire surface like in S. pneumoniae [31].
Localization of four GBS sortase A-substrates revealed very
different subcellular distributions that can be defined as uniform
(Alp2), polar (PilB and Gbs0791), or punctuated (CspA), as
compared to the septal distribution of SrtA. As LPXTG
containing proteins are covalently linked to the cell wall precursor
lipid II prior to being incorporated into the mature peptidoglycan
[6], one can expect a spatial proximity of the secretion machinery
(SecA) with the anchoring (SrtA) and peptidoglycan factories.
Streptococci which are elongated ellipsoid organisms called
ovococci [14] possess two cell wall synthesis systems. One is
associated with septum formation where PG is synthesized at the
cell division creating the septal wall required for formation of the
new poles; the other is a multi-protein complex required for the
slight longitudinal peripheral elongation of their sidewalls prior to
Figure 5. Surface distribution of Bsp in S. agalactiae NEM316and derivatives. Bacteria were harvested in exponential phase(OD600 = 0.3) and labeled with DAPI (blue) plus rabbit anti-Bsp pAb(green). The bacterial strains studied were: NEM316 (WT) (positivecontrol); NEM316Dbsp/pTCV-erm without insert (negative control);NEM316Dbsp/pTCV-erm expressing recombinant Bsp with the signalpeptide of CspA, Alp2, PilB, or Gbs0791. Data are representative of threeindependent experiments.doi:10.1371/journal.pone.0065832.g005
Figure 6. Surface localization of SrtA and unrelated cell wall-anchored proteins in S. agalactiae NEM316. (A, B) Bacteriaharvested in exponential phase (OD600 = 0.3) were labeled with (A)DAPI (blue) or guinea pig anti-SrtA pAb (green) and (B) with rabbit pAbagainst PilB, Gbs0791, Alp2, or CspA (red).doi:10.1371/journal.pone.0065832.g006
Figure 7. Surface distribution of newly synthesized Gbs0791 inS. agalactiae NEM316. Bacteria were harvested in exponential phase(OD600 = 0.3), incubated for 1 h with trypsin (0.1 mg/ml), washed twicein TH broth, and incubated at 37uC for 1 h and 2 h. Untreated ortrypsinized cells after various recovery times were labeled with DAPI(blue) or rabbit anti-Gbs0791 pAb (red).doi:10.1371/journal.pone.0065832.g007
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division. Indeed, fluorescent vancomycin, which is used to label
new peptidoglycan incorporation sites revealed both septal and, to
a lesser extent, sidewall localizations (Fig. 3). These observations
support the idea that LPXTG proteins are secreted by the SecA
machinery at the septum and then immediately anchored to the
peptidoglycan precursor by SrtA in S. agalactiae. In line with this
model, de novo synthesis of the LPXTG protein Gbs0791 after
trypsin digestion revealed that this protein is secreted at the
septum and that polar distribution most probably results from a
post-secretion process. We speculate that the different localizations
of LPXTG proteins at the bacterial surface likely depend upon
several factors: anchoring efficiency which itself depends on
protein synthesis levels and stability, protein association with the
membrane, protein diffusion rate in the lipid layer, but can also
depend on the activity of other LPXTGase as those identified in
both S. pyogenes and S. aureus [34,35]. We hypothesize that polar
localization of highly stable LPXTG proteins, like for example
PilB or Srr1 [36], could be the result of protein secretion and
anchoring at the septum, followed by redistribution through septal
and peripheral peptidoglycan synthesis [14] and accumulation to
the poles at later stages.
Surprisingly, the GBS capsular polysaccharide was found to
play an important role for the proper spatial localization of surface
proteins.
In conclusion, this study constitutes the first report showing the
spatial localization of surface proteins in the gram-positive
pathogen Streptococcus agalactiae. We provide evidence that secretion
and cell wall anchoring machineries are localized at the division
septum and that the YSIRK- signal peptide-dependent localiza-
tion rule did not apply to S. agalactiae. Our results contrast with
those reported previously in the close relative species of S. pyogenes
but the distribution pattern of SecA in the non-capsulated strains
of GBS and S. pneumoniae are similar. Thus, we conclude that the
localization of surface proteins in a given bacteria is highly specific
and probably depends on the organization of other cell surface
components such as capsule, extracellular polysaccharide, or
lipoteichoic acids.
Experimental Procedures
Bacterial strains and growth conditionsS. agalactiae NEM316 belongs to the capsular serotype III (ST-
23). The complete genome sequence of this strain has been
determined [7] and is accessible (NCBI RefSeq NC_004368.1).
The uncapsulated S. agalactiae NEM316 mutant was described
previously and referred to as cpsD mutant [27,38]. However, since
a change in the nomenclature of capsular genes, cpsD was renamed
cpsE [39]. Escherichia coli DH5a (Gibco-BRL) was used for cloning
experiments. S. agalactiae (GBS) and S. pyogenes (GAS) were cultured
in Todd-Hewitt (TH) broth or agar (Difco Laboratories, Detroit,
MI) and E. coli in Luria-Bertani (LB) medium. Streptococcus agalactiae
(GBS) and S. pyogenes (GAS) strains were cultured in Todd-Hewitt
(TH) broth in standing filled tubes without agitation. Erythromy-
cin was used at the following concentrations in mg/mL: for E. coli,
150 and for GBS, 10. All incubations were performed at 37uC.
General DNA techniquesGenomic DNA from S. agalactiae was prepared using the
DNeasy Blood and Tissue kit (Qiagen). Analytical PCR used
standard Taq polymerase (Invitrogen, Life technologies) and
preparative PCR for cloning and sequencing was carried out with
a high fidelity Phusion polymerase (Finnzymes). Plasmids for
overexpression (pTCV backbone) were constructed by standard
cloning procedures [40] and all inserts were entirely sequenced to
exclude mutations (GATC). All PCR primers used in this study
(Bsp translational fusions and histidine-tagged fusion proteins) are
listed in Table S1.
Rabbit and guinea pig polyclonal antibodiesThe sequences encoding CspA (residues 36 to 564 with a NH2-
terminal 66His-tag) and Gbs0791 (residues 33 to 361 with a
COOH-terminal 66His-tag) were cloned in pET28 whereas that
encoding SrtA (residues 47 to 237 with a COOH-terminal 66His-
tag) was cloned in pET26b. The corresponding histidine-tagged
fusion proteins were produced in E. coli BL21(lDE3) and purified
on nickel-nitrilotriacetic acid (Ni-NTA) agarose as described
(www.qiagen.com). Rabbit polyclonal antibodies (pAb) against
CpsA and Gbs0791 were obtained from Covalab (www.covalab.
com). Guinea pig polyclonal antibodies against SrtA were from
Eurogentec (www.eurogentec.com). Rabbit antisera against the
CAMP factor (Gbs2000) [23], the PilB pilin (Gbs1477) [24], and
the secreted protein Bsp (Gbs1420) [22], were from our antibody
collection. Rabbit polyclonal anti-Alp2 antibodies were kindly
provided by Gunnar Lindahl (University of Lund, Sweden).
Rabbit pAb directed against E. coli SecA were purchased from
IMBB (www.imbb.forth.gr/groups/minotech-new/antibodies.
html; ref 711-1). For double-labelling experiments, mice polyclonal
anti-PilB antibodies were obtained at the Institut Pasteur core
facilities as described [24].
Immunofluorescence analysesS. agalactiae NEM316 grown overnight in 10 ml of TH
(OD600<2) was diluted to get an initial OD600 of 0.05 (1/40
dilution) and grown at 37uC until OD600 reached 0.5 and re-
diluted again in TH (1/10) until reaching an OD600 of 0.5 and
diluted again (1/10) before final collection at mid-exponential
phase (OD600 of 0.3) to get a majority of exponentially growing
Figure 8. Conventional immunofluorescence microscopy show-ing localization of SecA in the non-capsulated mutant DcpsEcompared to the parental WT NEM316. (A) Immunofluorescencemicroscopy of bacteria harvested in mid-exponential phase andvisualized with fluorescent vancomycin (green) or plus rabbit anti-SecApAb (red). Note that SecA is more concentrated in the constrictingsepta and its neighboring region in a pattern very similar to thatreported for the non-capsulated strain of S. pneumoniae [31]. (B)Immunofluorescence of bacteria harvested in mid-exponential phaseand visualized with rabbit pAb against Bsp and PilB.doi:10.1371/journal.pone.0065832.g008
Immunolocalization of Surface Proteins in GBS
PLOS ONE | www.plosone.org 7 June 2013 | Volume 8 | Issue 6 | e65832
bacteria. Bacteria were washed twice in phosphate buffered saline
(PBS) before fixation in PBS containing 3% paraformaldehyde for
15 min at RT. For SecA detection, bacteria were pretreated with
lysozyme (1 mg/mL final concentration) for 15 min at 37uC and
then permeabilized with PBS-Triton X-100 (0.4%) for 5 min at
RT, washed twice with PBS and then fixed. Fixed bacteria were
washed twice with PBS and incubated for 45 min with rabbit or
guinea pig primary antibodies diluted in PBS-BSA 0.5% at the
following dilutions: Alp2 (1/1000), Bsp (1/200), PilB (1/300),
Gbs0791 (1/100), CAMP (1/50), SecA (1/200) and SrtA (1/100).
The specificity of Alp2 and PilB antisera has been tested previously
[8,23]. After three washings with PBS, samples were incubated for
30 min with secondary AlexaFluor 488- or AlexaFluor 594-
conjugated goat anti-rabbit immunoglobulin (1/300 dilution;
Molecular Probes, Invitrogen). Bodipy FL-vancomycin was
purchased from Molecular Probes and used at 3 mg/mL for
20 min at RT. Coverslips were mounted with 4 ml of Vectashield
mounting medium containing DAPI (Vector Laboratories, Inc).
Microscopic observations were done on a Nikon Eclipse E600 and
images acquired with a Nikon Digital Camera DXM1200F.
Immunofluorescence experiments for SecA and Bsp have been
performed at least 20 times independently and images have been
analyzed for about 800 bacteria appearing mostly in chains.
Immunolocalization of SrtA, PilB, Alp2, and CAMP were
performed at least 4 times independently and images are
representative of about 100 bacteria.
Imaging and image processingImage acquisitions of z-series in wide field fluorescence was
performed at the Imagopole on Zeiss Axiovert 200M epifluores-
cence microscope connected to a CCD camera. Images were
acquired with an apochromat 1006 (numerical aperture NA 1.4)
objective lens coupled to a Piezo system (Princeton Instruments)
and processed with Metamorph Software, version 6.1. Stacks of 10
images acquired every 300 nm in the z-axis were deconvolved
with the Metamorph Deconvolution module. Maximum projec-
tion of the deconvolved stacks of serial optical sections were
generated by selecting the highest intensity values along the z-axis
and displaying them as an XY.
ImmunoblottingFor analysis of SecA expression, total protein extracts were
loaded on SDS-PAGE and transferred to nitrocellulose mem-
brane. SecA was detected using rabbit-specific pAb and Horse-
radish peroxidase (HRP)-coupled goat anti-rabbit secondary
antibody (Zymed). Detection was performed with the Western
pico chemiluminescence kit (Thermo Scientific).
Supporting Information
Figure S1 Western blotting showing that the polyclonalantibody directed against SecA of E. coli recognizes aband of approximately 90 kDa in GBS despite a low levelof identity at the amino acid level. Western blot showing the
specificity of Alp2 antiserum on the cell wall extracts from WT
NEM316 and two transposon mutants inserted at two indepen-
dent sites in gbs0470 (alp2) (M1: NEM3695 and M2: NEM3694).
(TIF)
Figure S2 Low magnification image of SecA and SrtAlabeling in GBS strain NEM316. Single cocci displaying a
uniform labeling are indicated with white arrows.
(TIF)
Figure S3 An ExPortal-like domain enriched in anioniclipids can be visualized using the NAO (10-N-nonyl-acridine orange 1 mM) dye in GBS strain NEM316.
(TIF)
Table S1 List of oligonucleotides used in this study.
(DOCX)
Acknowledgments
We thank our former colleague Dr Lila Lalioui for the construction of the
NEM316 Dbsp mutant and the purification of the recombinant histidyl-
tagged Bsp protein. We thank Arnaud Firon for the isolation of two Himar
mutants (NEM3694 and NEM3695) in gbs0470 encoding Alp2. We thank
our colleagues from the Imagopole for their invaluable help in
deconvolution analyses. We thank the reviewers for their critical eye and
their useful suggestions to improve this study.
Author Contributions
Conceived and designed the experiments: SB PTC SD. Performed the
experiments: SB EC SD. Analyzed the data: SB PTC SD. Wrote the paper:
SD PTC.
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